Hi
My name is Julia. I am undertaking my PhD at the Peter Maccallum
Cancer Institute in Melbourne Australia. I am using PCR to amplify
mouse DNA using a variety of SSLP's obtained from the Whitehead
Institute. I am writing for help because all of a sudden my PCR has
gone from working beautifully to half working to not working at all. Here is my procedure. Please if anyone can give me any clues that would be great.
Because I am dealing with many tumour DNA samples at once, the
procedure I use is to make up a PCR mix. This contains everything
except the DNA. That is the primers, both unlabelled and labelled,
MgCl2, dNTP's, taq polymerase, polymerase buffer and H2O. I add the
DNA to the PCR mix. I then place the samples into the PCR machine
and do a 5 min. denature at 94C before starting the amplification
cycles. Usually what I see on a PAGE/formamide gel are 2 bands per
DNA sample since the DNA was isolated from heterozygous mice.
Occassionally I will see loss of heterozygosity which is what I am
looking for. Now what I am seeing on these gels is one band only.
Bizarre! It appears that the primers are preferentially annealing to
and amplifying one allele. However, when I run the 2 genomic DNA
controls I get amplification of one of them, the same one that
amplified in the tumour DNA samples but there is no amplification of
the other DNA control. What am I to make of this? Has something
happened to the DNA? Should I try a hot start? I await your reply.
Thanks.
Julia McKenzie
juliam at ariel.ucs.melbuni.edu.au